According to swine TIGAR mRNA sequences available in GenBank （No：XM_001926543）, a pair of primers was designed, with experimental condition optimizing, the method of a Real-time fluorescent quantitative reverse transcription-polymerase chain reaction （RT-PCR） was established to detect swine TIGAR gene, and the level of TIGAR mRNA of Landrace in different slaughter time was compared with the method established.The results showed that the R² of standard curves of TIGAR and the housekeeper gene β-actin were 0.9995 and 0.9996, respectively, and the amplification efficiency of TIGAR （103.7%） was consistent with β-actin （104.1%）.The methods established can be used for the determination of TIGAR mRNA in swine with high specificity and sensitivity. The research result was valuable to application.

To identify the possible role of very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) genes in duck muscles, the developmental changes of VLDLR and ApoER2 mRNA level in breast muscle, leg muscle and cardiac muscle during duck early growth stages were investigated using Real-time fluorescent quantity PCR. The results showed that VLDLR and ApoER2 mRNA expression level was different in different muscle tissues from 1 to 8 week in duck and had tissue-dependent. Three expression patterns of VLDLR mRNA during growth were found: Fluctuating expression as up-down (breast muscle), as down-up-down-up (leg muscle) and as down-up-down (cardiac muscle). For ApoER2 mRNA expression level, in breast muscle, the mRNA expression level was the highest (P<0.01) in 3 week old duck. In leg muscle, the mRNA expression level kept a low level in the first 6 weeks, then had a significant increase in 7 and 8 weeks, which was significantly higher than that of other weeks (P<0.01). During the cardiac tissue development, the mRNA expression level presented as up-down with growing, and had a higher expression level in the first 2 weeks (P<0.01). In the same week VLDLR and ApoER2 mRNA expression level had tissue-dependent in muscles of three different sites in duck. Our results indicated that VLDLR and ApoER2 genes could play an important role in intramuscular fat accumulation with spatial and temporal specificity during duck early growth stages.

We designed and synthesized two pairs of specific primers amplified S1 gene by RT-PCR method to investigate the variation in S1 genes of porcine epidemic diarrhea virus (PEDV) in 2012 in Henan.S1 genes of 5 PEDV strains were cloned and sequenced, and their phylogenetic trees were analyzed from different swine breeding farms. Sequences analysis showed that S1 genes shared 98.3% to 99.5% nucleotide identities and 97.1% to 98.9% amino acids homologies among five PEDV isolates. Compared with domestic landing PEDV from 2011, the nucleotide homologies were 88.6% to 98.0% and amino acid homologies were 85.3% to 98.7%. Compared with CV777, the nucleotide homologies were 88.7% to 89.1% and amino acid homologies were 87.3% to 88.4%. Homology analysis showed that S1 genes of 5 isolates shared the same genetic mutation, there were the same insertions and deletions. Compared with domestic mutant strains landed from 2011, there was no tendency. However, compared with CV777, there were three nucleotide insertions from 163 to 166 bp, nine nucleotide insertions from 173 to 174 bp, three nucleotide insertions from 405 to 406 bp and three nucleotide deletions from 463 to 464 bp. These insertions and deletions of nucleotides led to its corresponding changes in encoding amino acids. Phylogenetic analysis showed that S1 genes of 5 PEDV strains belonged to the third group. However compared with part of PEDV first group domestic mutant strains landed in 2011, the nucleotide homologies were 88.6% to 89.3% and amino acid homologies were 85.3% to 86.9%. CV777 was the second group. The results showed that S1 genes of PEDV prevalent strain exist in first and third groups, but mainly prevailing third group strains, the pathogenic and antigenic differences of these strains should be further studied.

The aim of this study was to develop a rapid method for the detection of Flavobacterium columnaris based on a double antibody sandwich ELISA （DAS-ELISA）. Purified monoclonal antibody against Flavobacterium columnaris was used as the capture antibody, while polyclonal antibody was used as the detection antibody. The optimal conditions for the ELISA were as follows: monoclonal antibody with 0.08 μg per well was added to coat overnight at 4 ℃; the plate was blocked by 30 g/L bovin serum albumin for 90 min at 37 ℃; incubation concentration of polyclonal antibody was 0.11 μg per well; the incubation time for detection antigen, polyclonal antibody and enzyme labeled antibody was 1 h at 37 ℃ for each; the value of D_{492 nm} was obtained after 15 min coloration. Judging with P/N≥2.1 and D_{492 nm}≥0.776 as positive criteria. This method had no cross reaction with Edwardsiella tarda, E. coli, Aeromonas hydrophila, Vibrio anguillarum, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio harveyi. Its minimum detectable limit was 1×10³ CFU. Therefore, this study provided a specific and sensitive detection method for Flavobacterium columnaris for the first time.  

To obtain the anti-kappa casein monoclonal antibody and complete the identification of the antibody characteristics. The BALB/c mice were immunized with kappa casein using foot-pad immunization. Popliteal lymph node cells from the immunized mice were fused with SP2/0 myeloma cells in the presence of PEG. Three hybridoma strains (1C4, 3G3, 3E6) which secreted the antibody specific for kappa casein were obtained．The sub-class of the antibodies were IgG1. The ascites were purified by Protein G affinity layer absorption column. The antigenic epitope of 3G3 and 3E6 were different and it was close between 1C4 and 3E6.The titer of purified ascites(1C4) was 1.28×10⁶ and the affinity constant was 2.89×10⁸ mol/L. A anti-kappa casein monoclonal antibody with good affinity had been achieved，which provided foundations for the rapid detection of casein in bovine milk samples.

In this study, a dual-priming oligonucleotide (DPO)-based PCR method for specific detection of Shigella was established using ipaH gene of Shigella as the target gene. The results showed that detection sensitivity of the DPO-PCR method was 1.65×10² CFU/mL. Compared with conventional PCR primers, the DPO primers were easily designed, which simplified the design procedure of PCR primers. DPO primers were not sensitive to annealing temperature, which could efficiently amplify the target gene in the annealing temperature range from 50 to 70 ℃. Moreover, due to the special structure of DPO primers, it had a higher specificity than conventional PCR primers, and none nonspecific amplifications were produced in reaction. In the practical application, tests on 133 samples including frozen/fresh meat, fruits and vegetables, fresh milk, eggs and cooked food by the DPO-PCR method showed that 15 samples were Shigella positive, which were in accordance with the testing results using GB method (GB/T 4789.5—2012), showing good practicality and reliability. The DPO-PCR method provided a new tool for fast and accurate detection of Shigella.

Two strains of porcine reproductive and respiratory syndrome viruses (PRRSV) were isolated from serum of some pig farms in Guangdong province and showed PRRSV positive in RT-PCR testing. The two viruses could passage stably and cause typical cenotaphic effect, they were named as LZ-GD and LB-GD. The analysis of variable region sequences of ORF5 and Nsp2 of the two viruses showed that LZ-GD and LB-GD strains were far to Europe strain Lelystad, the homology of nuclear nucleotide sequence were 63.5% and 63.8%, respectively, with classic American strain VR-2332 were 88.7% and 89.1%, respectively, and that with highly pathogenic JXA-1 strain were 99.2% and 99.3%, respectively. There were 30 amino acids deletion in Nsp2. It shared the deletion with JXA-1, HUN4 and other pathogenic variant. Thus, the two strains of PRRSV belonged to highly pathogenic American type.

In this study, Cashmere goat skin by extracting total RNA, cloning keratin-associated protein 1.1 (KAP1.1) gene eukaryotic expression vector pEGFP-N3-KAP1.1, using liposome-mediated recombinant plasmids were transformed into Inner Mongolia Cashmere goats cultured fibroblasts, temporal and spatial expression of the genes in eukaryotic cells, subcellular localization and expression pattern. The results showed that 2.0 μg plasmid and 6.0 μL ratio liposomes suitable for the optimization of the experimental protocol, KAP1.1 gene could be successfully expressed in fibroblasts in Inner Mongolia Cashmere goat, and 48 h after transfection, the transfection efficiency reaches a peak, the gene in eukaryotic cells in vitro on the nucleus, cytoplasm and cell membrane are expressed, but the strongest expression of the nucleus.

In this study, buffalo AQP8 gene was cloned and its eukaryotic expression vector was constructed, the expression pattern of AQP8 gene in buffalo ovary tissue was also assayed. The results showed that the CDS length of cloned buffalo AQP8 gene was 732 bp, and it shared 100% homology of amino acid sequence with cattle and mouse. AQP8 protein was detected in different developmental stages of buffalo follicles, it had significantly higher expression in the secondary follicles than that of in the primordial and the primary follicles (P＜0.05), and it mainly expressed in the granulosa cells of the secondary follicles. Clear EGFP green fluorescent was observed in transfected cell groups with transfection of the pEGFP-N1-AQP8 eukaryotic expression plasmid into HEK293T cells by Lipofectamine^R LTX and PLUS^TM reagent. The above results lay foundation to further investigate the function of AQP8 gene in the buffalo follicle development and granulosa cell apopotosis.

A fragment about 696 bp was amplified by PCR technique with specific primers based on Tams1 gene sequence（AF214842） of Theileria annulata reported in GenBank. Then the target fragment was directionally cloned into pGEX-4T-2 expression vector, the recombinant plasmid DNA was cut by enzymes, and then sequenced.The result showed that the homology of the cloned Tams1 gene was 98%. The positive plasmid was transformed into BL21（DE3）, and then induced by IPTG. Soluble fusion protein whose expression level reached 1.39 mg/mL was obtained when it was induced with 0.5 mmol/L IPTG for 4 h at 37 ℃, and it was 53 ku. Western blotting showed that recombinant protein GST-P27 which was purified by Glutathione Sepharose 4B had the favorable reactionogenicity.

The incompatibility plasmids of 101 strains Salmonella typhimurium were detected by PCR-based replicon typing. Eighteen pairs of primers were designed to perform 5 triplex-PCRs and 3 simplex-PCRs, recognizing FIA, FIB, FIC, HI1, HI2, I1, L/M, N, P, W, T, A/C, K, B/O, X, Y, F and FII. 94 strains (93.1%) Salmonella typhimurium carried a total of 10 Inc groups. The results suggested that the method was applicable for a large number of strains to trace the diffusion of specific multi-drug resistance plasmids in different environments.

This study was aimed to obtain expression of fusion gene （ORF2-TCE） of porcine circovirus type 2 (PCV2) in adenoviral expression system and develop PCV2 subunit vaccine. The target genes ORF2 and T cell epitope (TCE) were obtained by PCR with pMD18-T-ORF2 and pMD18-T-TCE as templates, respectively. The fusion gene ORF2-TCE was successfully obtained by overlap extension PCR method with the ORF2-linker and linker-TCE fragments as templates. The fusion gene was cloned into the transfer vector pShuttle-CMV. The PmeⅠ-linearized plasmid vector pShuttle-CMV-ORF2-TCE was transformed into Escherichia coli bacteria competent cell BJ5183, containing the pAdEasy-1 vector by electroporation. The recombinant plasmid named pAd-ORF2-TCE was obtained and identified by PCR and PacⅠenzyme digestion. To produce the recombinant adenovirus rAd-ORF2-TCE, AD293 cells were transfected with PacⅠ-linearized plasmid pAd-ORF2-TCE. The recombinant virus named rAd-ORF2-TCE were obtained and purified through vital plaque by three times and the viral titer was 10^12.3 TCID₅₀/mL. The expression of the fusion protein was verified by Western blotting and indirect immunofluorescence assay (IFA).

Peste des petits ruminants virus (PPRV) and goat pox virus (GTPV) are the causative agents of two kinds of goats’ diseases—peste des petits ruminants and goat pox which can cause disaster economic losses. In order to detect the two viruses simultaneously and quickly, two sets of primers and relative probes were designed based on the nucleoprotein (N) gene of PPRV and the inverted terminal repeat (ITR) segment of GTPV， respectively. In order to work together in the same reaction, the probes were labeled with different fluorescent materials 5′FAM-TAMRA3′and 5′JOE-Eclipse3′，respectively. Results showed that the duplex Real-time RT-PCR assay was identified to be specific for PPRV and GTPV only and specific fluorescent signal could be detected, but the related viruses including fowl pox virus（FPV）and canine distemper virus (CDV) had no specific fluorescent signal. Positive recombinant plasmids (PPRV pMD18-T-N and GTPV pMD18-T-ITR) were built and used for positive quantitative templates to establish duplex standard curves. The developed assay based on the probe N-ITR was found to be highly specific and sensitive with a detection limit of 10² copies/μL cDNA and 10³ copies/μL DNA for PPRV and GTPV， respectively. Finally, the duplex Real-time RT-PCR assay for simultaneous detection of PPRV and GTPV was established preliminarily in the study.

The protein encoded by E₀ gene is one of the major protective antigens of bovine viral diarrhea-mucosal disease virus (BVDV). E₀ gene is a candidate for developing genetic engineering vaccine of BVDV and it could not express in BCG, it has been shown that E₀ protein has RNase activity, and has a plurality of rare codons according to analyze the codon usage frequency of BCG. By truncating the gene and optimizing its codons, E₀ gene expressed in BCG successfully and its antigenic activity was detected, which laid the foundation for building bivalent genetic engineering vaccine to prevent tuberculosis and bovine viral diarrhea-mucosal disease at the same time.

The study was conducted to establish the duplex Real-time PCR assay for detecting both duck tembusu virus (DTMUV) and duck plague virus (DPV). According to the sequences of DTMUV E gene and DPV UL6 gene in GenBank, two sets of specific oligonucleotide primers for DTMUV and DPV along with two TaqMan probes were designed. The duplex Real-time PCR assay was developed through optimization of reaction conditions and validation of specificity, sensitivity and repetitiveness of the method. The sensitivity of the assay were both 100 template copies for DTMUV and DPV. There was no specific bands of the same sizes were amplified from other duck pathogens, such as duck Newcastle disease virus, duck hepatitis virus, muscovy duck parvovirus, duck circovirus, H9 subtype avian influenza virus, egg drop syndrome virus. This duplex Real-time RT-PCR assay is a sensitive, quick, specific and quantitative test for detection of DTMUV and DPV, and will be useful for the control of these viruses in ducks.

To investigate the effect of essential amino acids on LAT1 expression in lactation mammary gland, the lactation mammary acini were cultured and LAT1 and 4F2hc expression were detected by Western blotting. The results showed that essential amino acids upregulated LAT1 and 4F2hc expression significantly in lactation mammary acini of dairy cow. It revealed that LAT1 was the mainly amino acid transporter in lactation mammary gland. The expression of LAT1 and 4F2hc was induced by essential amino acids.

One nephropathogenic infectious bronchitis virus （IBV） strain was isolated from Qingdao city, named as QD isolate.S1 gene of the strain was amplified, cloned and sequenced. The S1 gene of QD isolate was composed of 1620 nucleotides, and a spike glycoprotein cleavage recognition site was Arg-Arg-Phe-Arg-Arg. The nucleotide acid similarities among the nine IBV vaccine strains and the QD strain were 78.8% to 82.2%. Phylogenetic analysis based on the S1 genes showed that QD strain and the vaccine strains belonged to different clusters, and showed larger evolutionary distances, but showed the smaller evolutionary distances with the field nephropathogenic IBV strains in China. The result showed that nephropathogenic IBV strains were widely popular in China, and the QD strain could be used as the infectious bronchitis vaccine candidate strain.

The assay was aimed to establish a rapid, sensitive and specific method of loop-mediated isothermal amplification (LAMP) for the detection of Salmonella. According to the highly conserved STN gene of Salmonella reported in GenBank, we designed a set of primers, and then followed by a series of LAMP optimization of reaction conditions, specificity test, sensitivity test, stability test and enzyme test. The sensitivity of LAMP and conventional PCR were compared.The results showed that the optimal reaction temperature of LAMP was 65 ℃, when only amplification of Salmonella, the minimum detectable concentration was 10¹ CFU/mL, which was more than conventional PCR detection by one order of magnitude. The results showed that LAMP method had the qualities of specificity, high sensitivity, short time-consuming and convenience for the detection of Salmonella, which was expected to develop into an effective method

A total of 28 Haemophilus parasuisc （H.parasuis） isolates were isolated from 48 farms in Longyan. Strains were confirmed by biochemical tests and PCR. Serovar 5 was the most prevalent, followed by serovar 4. 14 days old piglets were immunized with inactivated bacterin using seorvar 4 isolate LY04 and seorvar 5 isolate LY052, respectively, which were representative strains, and the results of efficacy test showed that the protection rates were both 66.7%.

Two pairs of PCR primers were designed according to sequence of canine distemper virus （CDV） and canine coronavirus （CCV） from GenBank, respectively, which could amplify 550 bp fragment for CDV and 225 bp fragment for CCV. The products of PCR were cloned to pMD18-T for sequencing, which proved to be specific. Positive plasma were developed for standard DNA, and the sensitivity result of duplex PCR showed that the method could amplify 0.1 ng/μL nucleic acid for both of viruses. The result of rudimentary application showed that the method was specific, sensitive, efficient, and was a new detecting method for the mixed infection of CDV and CCV.

In order to gain nucleocapsid protein （N） and glycoprotein （E） of porcine reproductive and respiratory syndrome virus （PRRSV）， the total RNA was extracted，ORF7 and ORF5 genes were obtained by RT-PCR， inserted into pET-28a(+) vector， and then transformed into Escherichia coli BL21（DE3），respectively. The expressed products were identified by SDS-PAGE and Western blotting， and purified by affinity chromatography. The results showed that N and E fusion protein were successfully expressed and the proteins had good reactionogenicities by Western blotting analysis. The purities of the purified proteins were 89% and 90%, respectively. This study could lay foundations for molecular biological function research and establishment of test methods for detection antibodies of PRRSV.

microRNA (miRNA) is a important endogenous non-coding single-stranded small RNA species,which play an important role in post transcriptional regulation of gene expression.The study on miRNA has become a focus in life science gradually in recent years. The importance of miRNA in regulating skeletal muscle development has been reported recently．This review summarized the researches on some key miRNAs related with skeletal muscle development.

In order to prepare the leptin receptor long form gene standard plasmid and standard curve using Real-time quantitative PCR, the total RNA was isolated from the arcuate nucleus tissue of ovis aries before puberty, and was synthesized the first-strand cDNA via reverse transcription.Using PCR technology, the leptin receptor long form gene was amplified.The aim gene fragment was recycled, and the fragment was then cloned into the pMD18-T plasmid vector.Recombinant plasmid of recycled products was constructed.After extraction and identification, the recombinant plasmids were quantified and the standard curve was established.The amplified products of the recombinant plasmids and secquence analysis confirmed that the pMD18-T-leptin receptor long form gene was successfully cloned.The standard curve showed high linearity, sensitivity, specificity and exercisable, which can be used to detect leptin receptor long form gene expression

Metagenomics is the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and laboratory cultivation of individual species and showing strong vitality in research of the metabolic pathway and exploring the resource of rumen microbiota. There are abundance of symbiotic microbiota in the gastrointestinal tract of animals, and they are deeply associated with the well-being of the host because of affecting normal physiology, metabolism and health, as well as the development of diseases. The application of metagenomics may solve many hot and difficult issues in the research of ruminant metabolism, especially camel metabolism. Therefore, this paper reviews the construction and selection procedures, especially introduced the Roche454 and Illumina high-throughput sequencing methods. Meanwhile, the application prospects of metagenomic in communities of microbial organisms are explored.

Using double-layer plate method, a phage against Salmonella enteritidis was isolated from sewage water, and its biological characteristics were studied. The plaques of isolated phage were transparent with clear boundary, and were about 2 mm in diameter at 12 h. The morphological properties of phage were examined by electron microscope, and showed that the phage had an isometric polyhedral no-enveloped head with diameter of about 54 nm, and a long noncontractile tail with size of about 110 nm. The test of optimal multiplicity of infection showed when MOI equaled 2, the number of phage offspring was higher. One-step growth kinetics was determined according to the results of one-step growth experiment, which showed that the latent period was about 10 min, the rise period was about 100 min, and the average bust size was about 61. The phage against S.enteritidis was successfully isolated from the sewage water in this study, which would provide materials for the further biological control researches of S.enteritidis using corresponding phage.

To seek out proteins which interact with structural proteins of porcine epidemic diarrhea virus （PEDV） in Vero E6 cells and study the mechanisms of viral infection.We created a Vero E6 cDNA yeast hybrid library. The total RNA of Vero E6 was extracted using Trizol method, and double strands cDNA was synthesized by SMART technique. We created a Vero E6 cDNA library by using homologous recombination in yeast. The results showed that the titer of cDNA library was 9.7×10⁸ CFU with the average of inserted fragments about 1.6 kb, and the recombination rate was 87%. These data indicated that the yeast two-hybrid cDNA library of Vero E6 was successfully constructed and might be useful for screening the host proteins interacting with structure protein of PEDV.

This paper aims at studying the microbial fermentation effect of two different bacterial inoculants, Bacillus subtilis and beer yeast, respectively, which were vaccinated on apple residue, then analying and comparing related influential conditions for mixed strains fermentation. Their true protein content in fermented materials were calculated through three factors orthogonal test level （size ratio, inoculation quantity, pH）.The results showed that the true protein content of mixed strains of Bacillus subtilis and beer yeast could reach up to 13.58%, and the biggest influenced factor was size ratio, followed by pH, recommending to fermentation parameters of size ratio at 1.0∶1, inoculation quantity of Bacillus subtilis with 3%, pH 5.The content of true protein may be further improved if yeast and bacteria agent was synergic at fermented apple residue.

This experiment was conducted to study the effects of different kinds and levels of acidifier on the fresh-keeping of feeds and diarrhea of growing mink. 100 male minks were randomly divided into 10 groups and 10 minks in each group.Ⅰgroup was fed basal diet.Ⅱ to Ⅹ groups were supplementation with different kinds and levels of acidifier in basal diet, that were 0.4% （Ⅱ）, 0.6% （Ⅲ）and 0.8% （Ⅳ） phosphoric acid, 0.5% （Ⅴ）, 1.0% （Ⅵ）and 1.5% （Ⅶ） citric acid, 0.5%（Ⅷ）, 1.0% （Ⅸ） and 1.5% （Ⅹ） lactic acid, respectively.The trial lasted for 62 days.The results indicated that acidifier could reduce the acid value （AV）, carbonyl value （COV） and total volatile basic nitrogen （TVBN） levels of fresh feeds, restrain the oxidation metamorphic process of fat and protein and improve the preservation ability of fresh feeds, and the 1.5% lactic acid was the best one.The number of Escherichia coli （37 ℃） were significantly affected by dietary acidifier, when tested the number of Escherichia coli at 1 hour, 1.5% citric group was the lowest one （P＜0.01）, at 4 h, the number of Escherichia coli was the lowest in 0.4% phosphoric acid group （P＜0.05）, at 8 h, the lowest Escherichia coli number was found in 1.5% lactic acid group （P＜0.05）.Supplementation with acidifier could extremely significantly reduce the acidifier index and diarrhea rate of growing minks （P＜0.01）, so it had a great significance to relieve the diarrhea of minks in summer.In conclusion, the best level of lactic acid was 1.5% in diet for minks.

Vitamin A （VA） is the essential nutrition factor as well as an important antioxidant for the animal, which has many functions, such as protecting epithelial integrity, promoting bone growth, regulating body metabolism and embryonic development and enhancing immune ability.However, recent studies have shown that VA acting as important immune adjuvant in the intestinal mucous membrane immunization and induction of lymphocyte apoptosis.This paper reviews the newest research progress on immune function of VA, so as to broaden the research area of nutrition and immunization.

Fat is an essential element for animal.The paper reviewed recent research progress on fat nutrition in monogastric animal at home and abroad, combining with the fat digestion and absorbing form in animal body, and summarized biological function and application of fat in production, so as to provide theoretical basis for better utilization of fat in animal production and exploration its mechanism of action.

This paper summarized the current development and application of yolk antibody （IgY） as a new feed additive in pigs, cattle, poultry, dog, shrimp and silkworm in China. Pointing out the problems faced in the industrialization production, such as safety, stability, single, oral limitation and standard procedures.To accelerate the process of IgY technology industry in China, which has extensive promotion and application in animal production.

:A strain of highly cellulolytic decomposing bacteria was obtained by the congo red flat plate straining and shake flasking method screened from the ox dung which named N12. It had been identified as Bacillus which belonged to Bacillus pumilus sp. by morphologic characteristics observation，physiology and biochemical experiment and also 16S rRNA genetic analyze.The conditions of producing cellulase on strain N12 were also been studied in this test.When the contents of carbon and nitrogen sources were 0.5% maizena and 1% yeast powder，and also the nitial pH was 3.0，the fermentation temperature was 42 ℃,at the same time the fermentation time was 54 h，it had the highest enzyme activity.

In order to study biological characteristics of Tibetan Mastiff somatic cells and preserve genetic resource, two fibroblast cell strains from Tibetan Mastiff were established by tissue culture. In vitro culture cell morphology, hyperplasia ability and some biological characteristics were researched. Tibetan Mastiff cells in vitro culture appeared typical morphology of fibroblast cells, grew well and had strong hyperplasia ability. Population doubling time of the cells was 46.1 h. Vimentin expression in the cells was confirmed by immunofluorescence staining. The rate of F5 cells with normal diploidy (2n=78) was 91.4%. Moreover, longer X chromosome belonged to submetacentric chromosome, but Y chromosome did telocentric chromosome. Tibetan Mastiff cell strains eslablished in the study offered the materials for the Tibetan Mastiff somatic cell nuclear transfer research.

In order to investigate the effect of glucose on oxidative stress in cultured goose primary hepatocytes, partial gene sequences of GPX1 and SOD1 genes were cloned, and the goose primary hepatocytes were treated with different concentrations（0（control group）, 5, 25 and 35 mmol/L） of glucose for 48 h, and then enzyme activities of GPX and SOD and mRNA levels of GPX1 and SOD1 were examined. The results showed that goose GPX1 gene sequence had the highly similarity with Zebra Finch, and SOD1 had the highly similarity with Red Jungle fowl. Compared with the control group, 5 mmol/L glucose had no significant effect on GPX enzyme activity （P>0.05）, but 25 and 35 mmol/L glucose could significantly decrease GPX enzyme activity (P＜0.05).5 mmol/L glucose could significantly increase SOD enzyme activity （P＜0.05）, but 25 and 35 mmol/L glucose had no significant effects on SOD enzyme activity（P＞0.05）.35 mmol/L glucose could significantly increase mRNA expression of GPX1 and SOD1 genes （P＜0.05）, but 5 and 25 mmol/L glucose had no significant effects on the two genes（P＞0.05）.In conclusion, the high concentration glucose （35 mmol/L） could induce intracellular oxidative stress in goose primary hepatocytes.

The assay was aimed to study the effect of Hedyotis diffusa Willd superfines powder on immune function. Three groups of mice were gavaged with the drugs at doses of 1.75,1.25 and 0.75 g/kg once a day for 7 consecutive days, repectively. The mice of control group were administered with the same volume of saline.At the last time, mice were injected into the muscle with Newcastle disease vaccine,and get peripheral blood on days 7, 14 and 21 after the medicine administration. The enhancement of innate immune responses were evaluated by using CCK-8 method, hemagglutination inhibition test, macrophage phagocytic function test and organ coefficient method. The levels of lymphocytes proliferative capacity, serum antibody, peritoneal macrophage phagocytosis and the indexes of immune organs in mice were extremely significantly enhanced (P＜0.01). Hedyotis diffusa dWilld superfines powder could extremely significantly regulate the immunity function of mice (P＜0.01).

The experiment was aimed to study the effects of different concentrations of daidzin and genistin on proliferation of goat mammary gland epithelial cells, the test was adopted the method of MTT. The results revealed that the ng level of concentration had more effects on cells than the μg level of concentration. And when cells were treated with the two materials for 48 h, the effects were greater than for 24 and 72 h. The highest poteney was detected in daidzin (10 ng/mL) and genistin (10 and 100 ng/mL) compared with the control group.  

It is of great importance to learn about the changes of reproductive tract mucosal immune status by studying the normal development of hens, especially during the immediate post-hatch and laying period, because the chicken reproductive tract associated lymphoid tissue （RTALT） is the first immune barrier of the genitourinary tract against the invasion of external pathogenic microorganisms. This study can serve as a theoretical basis of the reproductive tract disease prevention (especially salmonellosis) for laying hens, and ultimately achieve the goal of providing pollution-free eggs for people to consume. Therefore, this article on chicken reproductive tract associated lymphoid tissue structural basis for development as well as their immune function were reviewed.

Pulmonary surfactant protein （SP） is a carrier which plays an important role in phospholipids, mainly in the bronchial surface and alveolar gas-liquid interface, with lower surface tension properties, prevents alveolar collapse, is beneficial to gas exchange and to maintain the lungs pulmonary residual volume and to keep pulmonary compliance. Four kinds of proteins have been found, namely SP-A, SP-B, SP-C and SP-D. These four proteins in lung disease defensive and in maintaining lung function play important roles. Therefore, this article reviews SP from its structure, function and the correlation with the disease, to provide a basis for future research.

To investigate and assess the aggressive behavior phenotypic spectrum of Turpan gamecock and SNP of their behavior related genes, this study adopted the methods including mirror fighting and pair fighting to observe aggressive behavior phenotypes of 12 adult Turpan gamecock, and PCR-SSCP analysis about monoamine oxidase A (MAOA) gene in exons 1 and 2 of 103 Turpan gamecock and 71 New Romain chicken. The results showed that both methods of fighting strength of aggressive behavior phenotype sort basically the same, indicating that the behavior of aggressive behavior spectrum could be used for the preparation of aggressive behavior in Turpan gamecock fighting strength of phenotypic assessment. MAOA1 polymorphism in Turpan gamecock and New Romain was no difference after detection, but on the MAOA2, the polymorphism was not entirely consistent, suggesting MAOA2 might be had a certain correlation with Turpan gamecock aggressive behavior.

The SNPs of Yanbian Yellow cattles thyroglobulin (TG) gene were detected by cloning and sequencing, confirmed by technique of PCR-RFLP,and studied the association of polymorphisms of TG gene with growth traits. The results showed that there were C218T and A430G two mutations in the exon 48 in TG gene of Yanbian Yellow cattle. The association analysis showed that the chest breadth with TT genotype in C218T locus was significantly higher than that of CT genotype（P＜0.05）. A430G locus polymorphism significantly associated with the body weight, rump length and hucklebone width（P＜0.05）. The body weight of GG genotype was higher than AA genotype（P＜0.05）, the rump length of AA and AG genotypes were higher than GG genotype（P＜0.05）,and the hucklebone width of AG genotype was higher than GG genotype within population（P＜0.05）.In a word, C218T and A430G SNPs might be a good DNA marker to be used in MAS for growth traits.

This study aimed to clone the heat shock protein 70 (HSP70) gene, analyze its structure, and reveal the distribution of SNP in Wenchang chicken and Beijing-You chicken. HSP70 gene was amplified by PCR. Then the gene mutation was identified by sequencing. Three mutations were detected in the HSP70 genomic fragment, one in the coding region (A-1044G) and two in the 5′regulatory region (T-564C, A-68G). PCR-RFLP was used to analyze the polymorphisms of T-564C locus in our experimental chicken populations. The frequency of C allele was 0.87 in Wenchang chicken, 0.55 in Beijing-You chicken. Bioinformatics analysis of the promoter region had revealed that some transcription factor Sp1, AP-1, GATA-1, CREB, MZF1 and HSF2 were represented in this region.

This experiment was aimed to study the effect of quinestrol on the expression of PCNA and Caspase-3 in spermatogenic cells of juvenile mice. 4-week old male mice were randomly divided into four groups. Mice in the control group were injected intraperitoneally with olive oil alone and mice in the experiment groups were injected intraperitoneally with octylphenol resolved in olive oil at 1, 10, 100 mg/kg doses respectively for 1 week (once a day). The testes were immediately removed for weighing and serial cross-sections that were used for histological examination when the mice were 8-week-old. The expressions of PCNA and Caspase-3 in spermatogenic cells were determined by immunohistochemical method. The results showed that the thickness of the convoluted seminiferous tubule decreased, cellular deranged, cell layers decreased and intercellular space increased. Meanwhile, there were a large number of abnormal shedding of spermatocytes and round spermatids, but few elongated spermatids and spermatozoa. The expression of PCNA extremely significantly decreased and the expression of Caspase-3 extremely significantly increased as quinestrol dose increased (P＜0.01). Quinestrol caused male reproductive function injury through inhibiting proliferation and promoting apoptosis in spermatogenic cells by regulating the expression of associated proteins.

In the long period of time, the research of cell differentiation process, especially for lineages of stem cells and the difference of their function, mainly focused on the regulation of gene transcription level. However, as increasing numbers of researches indicated that epigenetic mechanism （DNA methylation, chromatin remodeling, genomic imprinting, chromosome inactivation, non-coding RNA, etc.） has significant impact on the mammalian development, embryonic cells differentiation and proliferation of cancer cells, in which DNA methylation is the most common form. In this paper, research shows how DNA methelation impacts on transcription of DNA, molecular regulation, neuronal development and genetic.

Current cervical artificial insemination（CAI）procedure involving deposition of excessive sperm numbers is uneconomical for pig industry that is more outstanding in the use of high value-added semen product. In recent years, a deep insemination procedures in combination with fixed-time AI that was greatly developed and applied have shown to be beneficial for reducing the number of sperm required per pregnant sow, thus allowing the best use of valuable boars and, ultimately, the commercial integration of frozen-thawed and sexed sperm. This article provides an overview of the research development and application of the deep insemination procedure for liquid-stored, frozen-thawed and sexed sperm. Variables such as boar fertility, viable sperm rate, sow management, timing of semen delivery and insemination technology should be carefully considered to improving the fertility outcome.

Mink calicivirus is the member of caliciviridae. It can cause the mink hemorrhagic pneumonia and gastroenteritis. The disease even brook out in mink farming in Hebei and Shandong province. It has higher lethality. The virus had been isolated and identified from hemorrhagic pneumonia of mink by many scholars.This article presents a comprehensive review on progress in taxonomic position and biological characteristics, pathogenesis, disease prevention and control of it.

The purpose of this study was to explore whether moderate exercise could reduce inflammatory injury in lungs and prolong the survival of mice with influenza. The mice were divided into two groups (control and moderate exercise groups), control group’s mice were fed in the same condition as moderate exercise group, without treadmill exercise, the mice in moderate exercise group performed treadmill exercise (8 to 9 m/min, 30 min/d, 5 times/week for 2 weeks), all mice were nasally infected with FM1 influenza virus on day 14. On day 3 after being infected with the virus, five mice in exercise and control groups were killed for the lungs, two of which were for pathological analysis and three of which were for detection of IL-6, TNF-α, MCP-1 and virus titer. On day 3 after infected with the virus, there were lower inflammatory cells, virus titers and IL-6, TNF-α, MCP-1 （P＜0.05） in the lungs of mice in exercise group than that in control group, furthermore the survivals of mice in exercise group were also apparently longer than that in control group. Moderate exercise could significantly reduce the inflammatory injury in lungs of the mice with influenza and apparently prolong the survival of the mice.

Acinetobacter baumannii is an important condition opportunistic pathogen widely distributing in human living environment. Because of its strong drug-resistant character, it has always been a hot topic in the biomedical research field. However, little knowledge of its virulence factors is known comparing to the drug-resistance research. Acinetobacter baumannii can survive for a long time in host and cause serious damage to host cells after invading the host, so virulence factors might play an important role during the invasion, proliferation and injure process. Here, we summarized the advanced researches and progresses on virulence factors of Acinetobacter baumannii for benefiting future study of its pathogenic mechanisms, providing new ideas for the potential treatment and screening drugs of diseases caused by Acinetobacter baumannii.

In recent years, the epidemic intensity of porcine epidemic diarrhea virus (PEDV) have continuously enlarged,causing a huge economic losses in pig industry. This paper summarizes the research progress on the isolation and identification,variation, molecular biology of PEDV.This paper based on the current immunity results ot the PEDV vaccines, the research and development direction of PEDV production, with a view to the new, more useful product of PEDV.

The experiment was aimed to study the effects of different doses of Chinese herbal medicine on immune efficiency of Newcastle disease oil emulsion inactivated vaccine in laying hens. Two hundred and fourty 100-day-old Brown laying hens were randomly divided into 4 groups with 5 replicates in each group and 12 hens in each replicate.The control group was fed with the basal diet with no adding Chinese herbal medicine, groupsⅠ, Ⅱ and Ⅲ were fed with the basal diet supplemented with 0.5%, 1.0% and 1.5% Chinese herbal medicine, respectively.The results showed that the serum Newcastle disease antibody titer of laying hens and serum Newcastle disease maternal antibody titer of chicken in groups Ⅱ and Ⅲ were significantly higher than those in control group and groupⅠ（P＜0.05）, moreover, groups Ⅱ and Ⅲ, control group and groupⅠ both had no significant differences（P＞0.05）. It pointed out that adding 1.0% or 1.5% Chinese herbal medicine could increase the Newcastle disease antibody titer, enhance the body’s immune ability.

This paper researched antibacterial effect of different Bidens bipinnata L extracts against clinical ESBLs E.coli by means of micro-dilution assay and the present method of combining traditional Chinese herbs with western medicine in vitro. The results showed that it had good inhibitory effect under Bidens bipinnata L decoction, ethyl acetate extracts, acetone extracts and trichloromethane extracts in combination with antibiotic. And ethyl acetate extracts was the most efficient. Bidens bipinnata L decoction was the most widespread, and could reduce the MIC of different antibiotics.

Cefovecin is a long-acting, third-generation cephalosporin antibiotic，and has good pharmacokinetic characteristics．It can be quickly absorbed，reached the Cmax, and be slowly eliminated．Moreover，cefovecin is a new extended-spectrum semisynthetic cephalosporin with a single injection providing a complete 14-day course of therapy．Cefovecin can be extensively used in veterinary clinic．This review details its newest research status，mechanism of action，antibacterial activities in vitro and in vivo, and clinical applications.

Rhodomyrtus tomentosa is a wild medicinal plant, which has potential value of exploitation as a new veterinary drug. A review of current development in research concerning the chemical components, biological activity and clinical application profiles of Rhodomyrtus tomentosa is presented, and certain related problems are discussed to provide the theoretical basis for further application of Rhodomyrtus tomentosa.

The Newcastle disease inactivated vaccines were prepared with oil adjuvants respectively from domestic and import.The experiments were done in order to compare the new domestic oil and Marcol-52 according to a series of testing indexes. The results showed that the quality of the new domestic oil was better than Marcol-52, except the absorptive quality.

The objective of this study was to investigate the epidemic of three viral diarrhea diseases of yaks in northwest Sichuan province and provide certain scientific basis for controlling this kind of disease. 1070 yak serum samples from 8 counties of Aba state in northwest Sichuan province were detected by enzyme-linked immunosorbent assay (ELISA) to investigate prevalence of BVDV, BCV and BRV. The results showed that the average positive rates of BVDV, BCV and BRV antibody were 44.3%, 84.1% and 94.4%, respectively. The BVDV, BCV and BRV of yaks were widespread in northwest Sichuan province, further comprehensive prevention and control measures of viral diarrhea diseases should be strengthened in this region.

As high-yielded and good quality economic crops, sunflowers are widely grown in Hetao area in Inner Mongolia.While produced during the sunflower seed oil processing,sunflower seed meals,receptacles,stalks and other by-products are often fed on livestock,which would not only avoid the environmental pollution caused by the by-products stacking and burning, but also to make up for the lack of feed resources.By studying the general situation of sunflower cultivation in Hetao area in Inner Mongolia, and its by-products development and utilization as feed resources, the author has identified some problems during the research and tries to put forward corresponding solutions.

The assay was aimed to optimize the extraction process of the compound Epimedium by orthogonal experimental design. The extraction process of the compound Epimedium was optimized with water volume, extraction times and extraction time as independent variables. The extraction ratio of icariin was choosed as an evaluation index. The optimized extraction conditions were as follows, extracted in 10 times water (solid-liquid rate at 1∶10) for 3 times and heated for 1.5 h per time. These results indicated that the optimum extraction technology by the orthogonal experimental design provided experimental bases for improving the content of icariin and its industrial production.

Brucellosis caused by Brucella is a zoonotic infectious disease, and not only the serious influence to the healthy development of animal husbandry, but also to human health and public health security that exist a lot of threats.To the current prevention and control measures for animal vaccination, domestic and foreign existing multiple weak vaccines, each vaccine has its advantages and disadvantages, there is no vaccine for human currently, the laboratorys have used genetic engineering and other technical means to actively develop new vaccines, such as recombinant attenuated vaccines and marker vaccines, DNA vaccines and so on, this paper introduces the research status of new vaccines.

Urea is widely used in dairy industry as the most common non-protein nitrogen.It is important to use urea as protein feed，reducing the cost of feed and improving the production efficiency in dairy industry. This paper reviewed the study of urea used in dairy industry and the prospects of urea application，which provided a help for producter in dairy industry.